Lung injury is one of the major concerns for chest cancer patients that undergo radiotherapy as well as persons exposed to an accidental radiological event. Reduction/oxidation (redox) system plays a key role in lung injury via chronic upregulation of pro-oxidant enzymes. NOX2 and NOX4 are two important reactive oxygen species generating enzymes that are involved in radiation toxicity in some organs such as the bone marrow. In this study, we aimed to evaluate the expression of NOX2 and NOX4 signaling in rat’s lung tissues. Upregulation of these genes may be involved in radiation-induced lung injury. Moreover, we evaluated the role of pre-treatment with melatonin on the expression of these genes. Twenty male rats were divided into 4 groups as control; melatonin treated; irradiation; and irradiation with melatonin pre-treatment. Rats were exposed to 15 Gy 60Co gamma rays and sacrificed after 10 weeks for evaluation of NF-κB, TGFbR1, SMAD2, NOX2, and NOX4 genes expression by real-time PCR. Results showed the upregulation of all five genes. The expression of NOX2 was more obvious compared to other genes. Administration of melatonin before irradiation could attenuate the expression of all mentioned genes. Results indicate that upregulation of NADPH oxidase genes such as NOX2 and NOX4 may be involved in the late effects of lung exposure to ionizing radiation. Melatonin via downregulation of these pro-oxidant genes is able to attenuate radiation toxicity in the lung.